Deciphering complex protein interactions and regulatory networks is of central interest in understanding cellular function and development, and in elucidating the mechanism of disease. To this end, chemical biology has emerged as a powerful technique for dissecting these networks through the controlled perturbation of protein function using biologically active small molecule compounds. However, conventional chemical biology studies provide only population averaged measurements of large number of cells. This ensemble averaging conceals the ever-present heterogeneity in cellular response, and obscures the underlying biology. Furthermore, current technologies provide only crude means of modulating the chemical environment over time, making them poorly suited to the study of kinetics and of response to sequential stimuli. Deciphering the complex underlying networks and cell-cell variations manifest in cellular response requires new technologies for the quantitative and time-resolved analysis of single cells under well-defined and time-varying chemical environments. We will combine state-of-the-art techniques in microfluidics and microscopy to build a single-cell chemical biology platform for conducting highly parallel studies of the response of single cells to time-varying sequences of chemical conditions and bioactive small molecules. This fully automated platform will advance the state-of-the-art in single cell analysis and should find broad applicability to the study of many cell types. This new technology will be validated in the quantitative analysis of cell-cell heterogeneity, kinetics, and robustness arising in the yeast pheromone/filamentous growth pathway which serves as an archetypical model for studying the highly conserved MARK signal transduction conduits. Relevance to public health: A new instrument will be built to enable the analysis of single cells exposed to precisely defined and time- varying chemicals conditions and drugs. By allowing for experimentation at the single cell level, this instrument will improve both our understanding of, and our ability to treat, disease. Furthermore, the ability to precisely manipulate and interrogate single cells will enable new lines of inquiry in relevant health fields including cancer research, stem cell research, and developmental biology.